Showing papers on "Histone binding published in 2011"

A Ruthenium Antimetastasis Agent Forms Specific Histone Protein Adducts in the Nucleosome Core

Abstract: Keywords: bioinorganic chemistry ; cancer ; histone binding ; nucleosomes ; Rapta-C ; ruthenium ; Mass-Spectrometry ; Anticancer Metallodrugs ; Complexes ; Chromatin ; Binding ; Dna ; Particle ; Chemotherapy ; Selectivity ; Inhibition Reference EPFL-ARTICLE-171621doi:10.1002/chem.201100298View record in Web of Science Record created on 2011-12-16, modified on 2017-05-12

Structural and histone binding ability characterizations of human PWWP domains.

Abstract: Background The PWWP domain was first identified as a structural motif of 100-130 amino acids in the WHSC1 protein and predicted to be a protein-protein interaction domain. It belongs to the Tudor domain 'Royal Family', which consists of Tudor, chromodomain, MBT and PWWP domains. While Tudor, chromodomain and MBT domains have long been known to bind methylated histones, PWWP was shown to exhibit histone binding ability only until recently. Methodology/principal findings The PWWP domain has been shown to be a DNA binding domain, but sequence analysis and previous structural studies show that the PWWP domain exhibits significant similarity to other 'Royal Family' members, implying that the PWWP domain has the potential to bind histones. In order to further explore the function of the PWWP domain, we used the protein family approach to determine the crystal structures of the PWWP domains from seven different human proteins. Our fluorescence polarization binding studies show that PWWP domains have weak histone binding ability, which is also confirmed by our NMR titration experiments. Furthermore, we determined the crystal structures of the BRPF1 PWWP domain in complex with H3K36me3, and HDGF2 PWWP domain in complex with H3K79me3 and H4K20me3. Conclusions PWWP proteins constitute a new family of methyl lysine histone binders. The PWWP domain consists of three motifs: a canonical β-barrel core, an insertion motif between the second and third β-strands and a C-terminal α-helix bundle. Both the canonical β-barrel core and the insertion motif are directly involved in histone binding. The PWWP domain has been previously shown to be a DNA binding domain. Therefore, the PWWP domain exhibits dual functions: binding both DNA and methyllysine histones. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1.

Bromodomain-peptide displacement assays for interactome mapping and inhibitor discovery

Abstract: Histone lysine acetylation is a key component of epigenetic regulation of gene transcription. Bromodomains, found in histone acetyl transferases and other chromatin-associated proteins, bind selectively to acetylated lysines, acting as “readers” of the histone code, and have recently been shown to contain a druggable binding pocket. Here we report the development of high-throughput assays that quantify the binding of bromodomains to acetylated histone peptides. We have used these assays to screen for histone binding partners of as yet uncharacterized bromodomains, adding to current knowledge of the histone code and expanding the repertoire of assays for chemical probe discovery. We have also demonstrated that these assays can be used to detect small molecule binding from the very weak to the nanomolar range. This assay methodology is thereby anticipated to provide the basis both for broader interactome profiling and for small molecule inhibitor discovery.

Cooperative DNA and histone binding by Uhrf2 links the two major repressive epigenetic pathways.

Abstract: Gene expression is regulated by DNA as well as histone modifications but the crosstalk and mechanistic link between these epigenetic signals are still poorly understood. Here we investigate the multi-domain protein Uhrf2 that is similar to Uhrf1, an essential cofactor of maintenance DNA methylation. Binding assays demonstrate a cooperative interplay of Uhrf2 domains that induces preference for hemimethylated DNA, the substrate of maintenance methylation, and enhances binding to H3K9me3 heterochromatin marks. FRAP analyses revealed that localization and binding dynamics of Uhrf2 in vivo require an intact tandem Tudor domain and depend on H3K9 trimethylation but not on DNA methylation. Besides the cooperative DNA and histone binding that is characteristic for Uhrf2, we also found an opposite expression pattern of uhrf1 and uhrf2 during differentiation. While uhrf1 is mainly expressed in pluripotent stem cells, uhrf2 is upregulated during differentiation and highly expressed in differentiated mouse tissues. Ectopic expression of Uhrf2 in uhrf1−/− embryonic stem cells did not restore DNA methylation at major satellites indicating functional differences. We propose that the cooperative interplay of Uhrf2 domains may contribute to a tighter epigenetic control of gene expression in differentiated cells.

Crystal structure and function of human nucleoplasmin (Npm2): a histone chaperone in oocytes and embryos

Abstract: Npm2 is the mammalian ortholog of nucleoplasmin (Np), a well characterized histone chaperone in Xenopus oocytes and early embryos. 1,2 Nucleoplasmin 2 expression is limited to oocytes, where it accumulates in nuclei and is excluded from nucleoli. 3 In mouse embryos, Npm2 is present until the 8-cell stage and is then down-regulated. 3 In npm2−/− mice there is a reduced cleavage of fertilized eggs to the two-cell stage, which renders these females less fertile. In addition, the architecture of cell nuclei is altered in knockout cells, as nucleoli appear fragmented and chromatin compaction is adversely affected. These defects persist in early zygotes. 3 Thus, Npm2 may function as a histone chaperone to help remodel chromatin in oocytes and early embryos, 3 but this has not been demonstrated directly. Interestingly, Npm2 does not appear to be required for sperm chromatin decondensation 3, while Np plays an important role in this process in Xenopus eggs during fertilization.2 The general importance of histone chaperones is shown by the propensity of DNA to be damaged when nucleosome assembly is compromised and conversely, free histones may also be detrimental to cells.4 In Xenopus oocytes, Np may form storage complexes with H2A-H2B dimers while N1 associates with H3-H4 tetramers. Working in concert, these chaperones may direct nucleosome assembly in the early embryo. 5–7 Npm2 and Np have an overall sequence identity of ~46%. Both chaperones contain a conserved N-terminal domain (the core), followed by a C-terminal tail that contains two acidic tracts (denoted A2 and A3), with a bipartite nuclear localization signal located between them (Figure 1A). 8 Acidic tracts in Np are disordered and play a role in binding core histones 9–11 and linker histones. 12 This includes a short acidic loop in the N-terminal domain (the A1-tract), which may interact with sperm basic proteins 13,14 and histones. 9–11 Figure 1 Domain organization, sequence alignment and monomer structure of Npm2 Crystal structures have been determined for N-terminal domains of Xenopus Np and NO38 9,11,15 Drosophila NLP 10 and human Npm1. 16 This core domain is responsible for pentamer formation and in three of the crystal structures, decamers are formed by the face-to-face association of two pentamers. In each decamer, salt bridges are formed by conserved residues in the AKDE and K-loops of opposing subunits in the two pentamers. 9,11,16 Pentamers and decamers in the Np-family may each play a role in histone binding 9–11,16, but the precise mechanism is currently being debated. 17–19 In this paper, we present the crystal structure of a human Npm2-core pentamer. We then analyzed histone binding by Npm2-core and a longer version of the chaperone that contains a large acidic tract in the C-terminal tail (Npm2-A2). These studies show that hNpm2 requires the A2-tract to bind core histones. This is due to the fact that each A1-loop in Npm2 contains only a single acidic residue, which greatly reduces its electrostatic contribution to histone binding. We also made loop exchange mutants between Npm2 and Np to test the roles of the A1- and A2-tracts. On balance, we find the A1- and A2-tracts of Np may act synergistically in histone binding. We also show that Npm2 and Np form decamers when they bind the four core histones, while H2A-H2B dimers bind to a single pentamer. We then modeled an Npm2 decamer to reveal compensatory changes in residues of the AKEE and Q-loops of this chaperone. These residues may form hydrogen bonds between opposing pentamers when chaperone decamers are formed during histone binding. Finally, we present FRET and biochemical data to support a model in which H2A-H2B dimers bind directly to nucleoplasmins, followed by the addition of H3-H4 tetramers at higher radius to form larger complexes.

Structure and Histone Binding Properties of the Vps75-Rtt109 Chaperone-Lysine

Abstract: The histone chaperone Vps75 presents the remarkable property of stimulating the Rtt109-dependent acetylation of several histone H3 lysine residues within (H3-H4)2 tetramers. To investigate this activation mechanism, we determined x-ray structures of full-length Vps75 in complex with full-length Rtt109 in two crystal forms. Both structures show similar asymmetric assemblies of a Vps75 dimer bound to an Rtt109 monomer. In the Vps75-Rtt109 complexes, the catalytic site of Rtt109 is confined to an enclosed space that can accommodate the N-terminal tail of histone H3 in (H3-H4)2. Investigation of Vps75-Rtt109-(H3-H4)2 and Vps75-(H3-H4)2 complexes by NMR spectroscopy-probed hydrogen/deuterium exchange suggests that Vps75 guides histone H3 in the catalytic enclosure. These findings clarify the basis for the enhanced acetylation of histone H3 tail residues by Vps75-Rtt109.

Interaction with the histone chaperone Vps75 promotes nuclear localization and HAT activity of Rtt109 in vivo.

Abstract: Modification of histones is critical for the regulation of all chromatin-templated processes. Yeast Rtt109 is a histone acetyltransferase (HAT) that acetylates H3 lysines 9, 27 and 56. Rtt109 associates with and is stabilized by Nap1 family histone chaperone Vps75. Our data suggest Vps75 and Nap1 have some overlapping functions despite their different cellular localization and histone binding specificity. We determined that Vps75 contains a classical nuclear localization signal and is imported by Kap60-Kap95. Rtt109 nuclear localization depends on Vps75, and nuclear localization of the Vps75-Rtt109 complex is not critical for Rtt109-dependent functions, suggesting Rtt109 may be able to acetylate nascent histones before nuclear import. To date, the effects of VPS75 deletion on Rtt109 function had not been separated from the resulting Rtt109 degradation; thus, we used an Rtt109 mutant lacking the Vps75-interaction domain that is stable without Vps75. Our data show that in addition to promoting Rtt109 stability, Vps75 binding is necessary for Rtt109 acetylation of the H3 tail. Direct interaction of Vps75 with H3 likely allows Rtt109 access to the histone tail. Furthermore, our genetic interaction data support the idea of Rtt109-independent functions of Vps75. In summary, our data suggest that Vps75 influences chromatin structure by regulating histone modification and through its histone chaperone functions.

The ING tumor suppressors in cellular senescence and chromatin

Abstract: The Inhibitor of Growth (ING) proteins represent a type II tumor suppressor family comprising five conserved genes, ING1 to ING5. While ING1, ING2 and ING3 proteins are stable components of the mSIN3a-HDAC complexes, the association of ING1, ING4 and ING5 with HAT protein complexes was also reported. Among these the ING1 and ING2 have been analyzed more deeply. Similar to other tumor suppressor factors the ING proteins are also involved in many cellular pathways linked to cancer and cell proliferation such as cell cycle regulation, cellular senescence, DNA repair, apoptosis, inhibition of angiogenesis and modulation of chromatin. A common structural feature of ING factors is the conserved plant homeodomain (PHD), which can bind directly to the histone mark trimethylated lysine of histone H3 (H3K4me3). PHD mutants lose the ability to undergo cellular senescence linking chromatin mark recognition with cellular senescence. ING1 and ING2 are localized in the cell nucleus and associated with chromatin modifying enzymes, linking tumor suppression directly to chromatin regulation. In line with this, the expression of ING1 in tumors is aberrant or identified point mutations are mostly localized in the PHD finger and affect histone binding. Interestingly, ING1 protein levels increase in replicative senescent cells, latter representing an efficient pathway to inhibit cancer proliferation. In association with this, suppression of p33ING1 expression prolongs replicative life span and is also sufficient to bypass oncogene-induced senescence. Recent analyses of ING1- and ING2-deficient mice confirm a tumor suppressive role of ING1 and ING2 and also indicate an essential role of ING2 in meiosis. Here we summarize the activity of ING1 and ING2 as tumor suppressors, chromatin factors and in development.

Novel Histone H3 Binding Protein ORF158L from the Singapore Grouper Iridovirus

Abstract: Singapore grouper iridovirus (SGIV), a major pathogen of concern for grouper aquaculture, has a double-stranded DNA (dsDNA) genome with 162 predicted open reading frames, for which a total of 62 SGIV proteins have been identified. One of these, ORF158L, bears no sequence homology to any other known protein. Knockdown of orf158L using antisense morpholino oligonucleotides resulted in a significant decrease in virus yield in grouper embryonic cells. ORF158L was observed in nuclei and virus assembly centers of virus-infected cells. This observation led us to study the structure and function of ORF158L. The crystal structure determined at 2.2-A resolution reveals that ORF158L partially exhibits a structural resemblance to the histone binding region of antisilencing factor 1 (Asf1), a histone H3/H4 chaperon, despite the fact that there is no significant sequence identity between the two proteins. Interactions of ORF158L with the histone H3/H4 complex and H3 were demonstrated by isothermal titration calorimetry (ITC) experiments. Subsequently, the results of ITC studies on structure-based mutants of ORF158L suggested Arg67 and Ala93 were key residues for histone H3 interactions. Moreover, a combination of approaches of ORF158L knockdown and isobaric tags/mass spectrometry for relative and absolute quantifications (iTRAQ) revealed that ORF158L may be involved in both the regulation and the expression of histone H3 and H3 methylation. Our present studies suggest that ORF158L may function as a histone H3 chaperon, enabling it to control host cellular gene expression and to facilitate viral replication.

Stretching short sequences of DNA with constant force axial optical tweezers.

Abstract: Single-molecule techniques for stretching DNA of contour lengths less than a kilobase are fraught with experimental difficulties. However, many interesting biological events such as histone binding and protein-mediated looping of DNA1,2, occur on this length scale. In recent years, the mechanical properties of DNA have been shown to play a significant role in fundamental cellular processes like the packaging of DNA into compact nucleosomes and chromatin fibers3,4. Clearly, it is then important to understand the mechanical properties of short stretches of DNA. In this paper, we provide a practical guide to a single-molecule optical tweezing technique that we have developed to study the mechanical behavior of DNA with contour lengths as short as a few hundred basepairs. The major hurdle in stretching short segments of DNA is that conventional optical tweezers are generally designed to apply force in a direction lateral to the stage5,6, (see Fig. 1). In this geometry, the angle between the bead and the coverslip, to which the DNA is tethered, becomes very steep for submicron length DNA. The axial position must now be accounted for, which can be a challenge, and, since the extension drags the microsphere closer to the coverslip, steric effects are enhanced. Furthermore, as a result of the asymmetry of the microspheres, lateral extensions will generate varying levels of torque due to rotation of the microsphere within the optical trap since the direction of the reactive force changes during the extension. Alternate methods for stretching submicron DNA run up against their own unique hurdles. For instance, a dual-beam optical trap is limited to stretching DNA of around a wavelength, at which point interference effects between the two traps and from light scattering between the microspheres begin to pose a significant problem. Replacing one of the traps with a micropipette would most likely suffer from similar challenges. While one could directly use the axial potential to stretch the DNA, an active feedback scheme would be needed to apply a constant force and the bandwidth of this will be quite limited, especially at low forces. We circumvent these fundamental problems by directly pulling the DNA away from the coverslip by using a constant force axial optical tweezers7,8. This is achieved by trapping the bead in a linear region of the optical potential, where the optical force is constant-the strength of which can be tuned by adjusting the laser power. Trapping within the linear region also serves as an all optical force-clamp on the DNA that extends for nearly 350 nm in the axial direction. We simultaneously compensate for thermal and mechanical drift by finely adjusting the position of the stage so that a reference microsphere stuck to the coverslip remains at the same position and focus, allowing for a virtually limitless observation period.

Fuzzy logic point of view applied to diseases caused by dynamic mutations

Abstract: For the first time, this paper tries to explain the origins of diseases caused by unstable DNA repeats, known as dynamic mutations. It is common to find fairly simplified correlation between the number of DNA repeats and severity of diseases. By examining the case studies, it is possible to see that simplified model does not reflect real condition of the patients, so we have proposed more complex model which involves gender, immunological condition, age of onset and analysis of family tree. Conclusions are related to known molecular biology phenomenon such as DNA methylation, histone binding and RNA secondary structure.